The two primary objectives of this grant proposal are: A) A further refinement and definition of the cortical granule lectin hypothesis for establishing a block-to-polyspermy at fertilization in eggs of the amphibian Xenopus laevis. Three aspects currently represent major gaps in our understanding: 1) determination of the molecular characteristics of the lectin that relate its glycoprotein structure to its carbohydrate (galactose) binding activity. Structural investigations will include carbohydrate and protein sequence as well as the aggregation properties of the multimeric lectin. 2) The cortical granule lectin binds to its ligand in the jelly coat to form the F-layer which functions to block sperm penetration. The jelly coat ligand, which is itself a glycoprotein, will be isolated and physicochemically characterized. 3) At fertilization, the vitelline envelope is modified and converted into the vitelline envelope component of the fertilization envelope. This modification also blocks sperm penetration, as does formation of the F-layer. Two glycoprotein components of the vitelline envelope undergo reductions in molecular weight at fertilization. Biochemical definition of these changes and identification of the cortical granule agents (enzymes) responsible for these changes will be pursued. B) The second primary objective is to determine the potential universality of the lectin hypothesis for establishing a block-to-polyspermy at fertilization by investigating other biological test systems in an analogous way to what was previously done in Xenopus laevis. Specifically, we will use gametes of fish (the king salmon, Oncorhyncus tshawytscha) and a mammal (the domestic pig, Sous scrofa).